A major premise of this proposal is that RNAs present in biofluids represent a window into various cellular processes that exist in multiple tissues that release such RNAs. We have found that oncogenic KRAS mutations that occur in colorectal cancer can regulate miRNAs, mRNAs and long RNAs secreted from tumor cells in exosomes. This project (project 1) will determine what the mechanisms are by which these RNAs (cRNAs) enter the bloodstream using in vivo models and how these RNAs change with oncogenic mutations. This project will result in the creation of new tools to visualize and analyze such RNAs in real time and from blood samples in mouse models. We propose the following aims to address these questions. Aim 1. Determine circulating RNAs in human and mouse plasma that differ in normal individuals and those bearing colorectal tumors. We hypothesize that colorectal tumor cells secrete an altered composition of circulating RNAs (cRNAs) compared to normal cells. Further, many of these changes will be conserved from mouse to human CRC. The goal of this aim is to identify plasma cRNAs that are altered with tumor progression (advanced adenomas and carcinomas). Aim 2. Elucidate the pathway(s) by which tumor RNAs traffic into the circulation. Various mechanisms may contribute to CRC-induced cRNA alterations. In this aim, we will determine the route by which the exosome-associated miRNAs and long RNAs identified in Aim 1 enter the circulation. Aim 3. Determine the influence of tumor genotype on cRNAs using human primary tumor xenografts and determine the function of candidate trafficking proteins and interacting motifs in altering tumor RNA secretion. In conjunction with studies performed in Project 2, we will determine the mechanism by which RNAs are trafficked into exosomes and secreted into the blood. In this aim, we will determine how the genotype of human CRC xenografts affects exosome-associated cRNAs.